The present invention relates to a method for attaching or bonding a polyethylene glycol (PEG) compound to a macromolecule such as a protein, a carbohydrate or other polymeric material.
An early detailed description of the synthesis of polyethylene glycol derivatives has been given by Harris in JMS. Rev. Macromol. Chem. Phys. C25, 325, 1985.
The modification of proteins and carbohydrates by the attachment of polyethylene glycol (PEG) is known and is described, for example, by Abuchowski et al in J. Biol. Chem. 252, 3578, 1977. The process is often referred to as PEGylation.
Various coupling reactions between amino groups of proteins and carbohydrate molecules and the monomethyl ether of PEG equipped with an electrophilic functional group have also been described, see Zalipsky, Advan. Drug Del. Rev. 16, 157, 1995). The composition of the resultant graft copolymeric system is dependent on the number of available attachment sites on the starting polypeptide (or carbohydrate), the reactivity of the PEG reagent, the excess of such a reagent and the reaction conditions.
Francis et al. discloses a method of bonding polyethylene glycol to proteins and other macromolecules under very mild conditions by activation with tresyl chloride, see Biotechnol. Appl. Biochem. 12, 119, 1990.
Various methods of PEGylation are also described in U.S. Pat. No. 4,179,337, U.S. Pat. No. 4,732,863, U.S. Pat. No. 4,917,888, WO-86/04145, WO-90/04606, WO-90/06952, WO-90/13540, WO-91/01758, EP-400472, EP400486 and EP-183503.
The therapeutic value of polyethylene glycol modified proteins has also been reviewed, see Nucci et al., Advan. Drug Del. Rev. 6, 133, 1991 and Inada et al., J. Bioactive Compat. Polymer 5, 343, 1990.
We have developed a novel method of attaching a polyethylene glycol compound to suitably functionalized macromolecular materials. By a polyethylene glycol compound we include polyethylene glycol (PEG) itself and derivatives thereof (PEG derivatives) in which one or both of the terminal hydroxyl groups in the polyethylene glycol molecule has been previously modified.
According to the present invention there is provided a process for attaching a polyethylene glycol compound to a macromolecule to prepare a conjugate or adduct between the polyethylene glycol compound and the macromolecule which comprises the steps of:
(1) preparing an activated PEG or an activated PEG derivative by incorporating an acrylic ester, an acrylic thioester or an acrylamido group into the PEG or PEG derivative;
(2) reacting the activated PEG or PEG derivative with a macromolecular material comprising one or more sulphydryl groups, primary amino groups and/or secondary amino groups; and
(3) recovering the conjugate of the PEG or PEG derivative and the macromolecular material.
By a PEG derivative we mean a polyethylene glycol polymer in which one or both of the terminal hydroxyl groups found in polyethylene glycol itself has been modified. Examples of suitable modifications include replacing one or both hydroxyl group(s) with alternative functional groups, which may be protected or unprotected, with low molecular weight ligands, or with another macromolecule or polymer. Modification of the terminal hydroxyl groups in polyethylene glycol can be achieved by reacting the polyethylene glycol with compounds comprising complementary reactive. functional groups, i.e. functional groups which are able to undergo a reaction with the hydroxyl groups in polyethylene glycol.
Suitable PEG derivatives include compounds in which one of the terminal hydroxyl groups has been converted into a group having the formula ROxe2x80x94 in which R is an alkyl, cycloalkyl, aryl, aralkyl or alkaryl group. Preferably R is alkyl to give a terminal alkoxy group. Preferred alkoxy groups are C1-4 alkoxy, such as methoxy.
The macromolecular material may contain just sulphydryl, primary amino or secondary amino groups or it may contain a mixture of such groups. Suitable macromolecular materials for conjugation to a polyethylene glycol compound include therapeutic proteins such as interleukins, albumins, growth hormones, aspariginase, superoxide dismutase, monoclonal antibodies, as well as carbohydrates, such as starch and dextran. Many of these macromolecules are of biological origin. The macromolecular material may also be a polymer of biological origin which has been previously modified by a reagent to introduce a sulphydryl group or a primary or secondary amine group. A preferred macromolecular material is a protein or a poly(amino) sugar.
The preparation of a PEG or PEG derivative carrying an acrylic thioester group can be carried out using conventional techniques in which a PEG or PEG derivative containing a sulphydryl group is reacted with acryloyl chloride. The reaction is typically conducted in the presence of a base such as a tertiary amine, e.g. triethylamine, or an aqueous solution of sodium hydroxide at a temperature in the range of from 0 to 5xc2x0 C. to avoid radical polymerisation between the acrylic double bonds. Suitable solvents for the reaction include aliphatic and aromatic hydrocarbons, chloroform and other halogenated hydrocarbon solvents. (See J. March, Advanced Organic Chemistry, John Wiley and Sons, New York, 3rd Edition (1985), page 362.)
The preparation of a PEG or PEG derivative carrying an acrylic ester group can be carried out using conventional techniques in which a PEG or PEG derivative containing a hydroxyl group is reacted with acryloyl chloride. The reaction is typically conducted in the presence of a base such as a tertiary amine, e.g. triethylamine, or an aqueous solution of sodium hydroxide at a temperature in the range of from 0 to 5xc2x0 C. to avoid radical polymerisation between the acrylic double bonds. Suitable solvents for the reaction include aliphatic and aromatic hydrocarbons, chloroform and other halogenated hydrocarbon solvents. (See J. March, Advanced Organic Chemistry, John Wiley and Sons, New York, 3rd Edition (1985), page 346.)
The preparation of a PEG or PEG derivative carrying an acrylamido group can be carried out using conventional techniques in which a PEG or PEG derivative containing a primary or secondary amino group is reacted with acryloyl chloride. The reaction is typically conducted in the presence of a base such as a tertiary amine, e.g. triethylamine, or an aqueous solution of sodium hydroxide at a temperature in the range of from 0 to 5xc2x0 C. to avoid radical polymerisation between the acrylic double bonds. Suitable solvents for the reaction include aliphatic and aromatic hydrocarbons, chloroform and other halogenated hydrocarbon solvents. (See J. March, Advanced Organic Chemistry, John Wiley and Sons, New York, 3rd Edition (1985), page 370.)
An example of such preparations is reported by Bignotti et al. (Macromol. Rapid Communic. 15, 659, 1994). Acrylating agents other than acryloyl chloride may also be used, such as 1-acryloylbenzotriazole and N-acryloyloxysuccinimide. However, acryloyl chloride is the preferred acrylating reagent.
The activated PEG or PEG derivative can be characterized by Gel Permeation Chromatography (GPC), FT-IR and UV spectroscopy. The degree of activation can be determined by end-group titration which involves adding an excess of 2-mercaptoethanol and titrating excess thiol with a calibrated KI/I2 solution, or by UV spectroscopy, after calibration with a standard acrylamide or acrylic ester such as N-acryloylmorpholine or tetraethyleneglycol diacrylate, conducted at 233 nm.
Activated PEGs or activated PEG derivatives can be stored for several months at Txe2x89xa64xc2x0 C. in the presence of a desiccant. A radical inhibitor such as 4-methoxyphenol may also be added to prevent radical polymerization.
The reaction of the activated PEG or activated PEG derivative with the macromolecular material is preferably performed at pHxe2x89xa78, usually in the range 8 less than pH less than 9, in aqueous media. Below pH 7.5 the reaction rate tends to be slow. Alcohols or alcohol/water mixtures can also be used.
Although this step is generally conducted in the liquid phase, it is also possible to perform the reaction at the solid-liquid interface in the case of insoluble materials. When the macromolecular material is a solid, it should have sterically accessible sulphydryl, primary amine or secondary amine-groups.
Reaction temperatures in the range of from 15 to 60xc2x0 C., particularly 15 to 50xc2x0 C. can be used and typical reaction times are 12-48 hours. The preferred reaction temperature is between 20 and 30xc2x0 C. The occurrence of vinyl radical polymerization is inhibited by conducting the reaction under an inert atmosphere, in the dark and in the presence of a radical inhibitor such as 4-methoxyphenol.
Recovery of the conjugate that is formed is generally carried out by ultrafiltration in water or by evaporating the reaction solvent and extracting the crude material with an appropriate new solvent.
The choice of procedure is mainly governed by the solubility of the adduct and the starting materials, and by their molecular weight.
The purity of the adduct can be assessed by GPC, FT-IR, NMR and UV spectroscopy. Checking for residual activated PEG or PEG derivative is usually performed spectrophotometrically at 233 nm.
When a monoalkoxy, e.g. monomethoxy, PEG derivative containing a single sulphydryl, hydroxyl or amino group is used in the above process, as is preferred, such materials may often contain a small amount, typically less than 10% by weight, of a difunctional compound having two reactive sulphydryl, hydroxyl or amino groups. These difunctional compounds can be activated at both ends with acrylic double bonds to produce a compound which is able to react twice with the macromolecular material to produce a cross-link. This can be avoided, if desired, by modifying the standard process by conducting an additional processing step (2xe2x80x2) between steps (2) and (3). This additional step (when employed) comprises adding a molar excess, relative to the activated alkoxy PEG derivative, of a compound which is capable of adding to residual double bonds. Typical compounds include mercaptans, such as 2-mercaptoethanol, and secondary-amines, such as morpholine. The additional step (2xe2x80x2) is preferably performed under the same conditions as step (2), i.e. at a pHxe2x89xa78, usually in the range 8 less than pH less than 9, in aqueous media at a temperature in the range of from 15 to 60xc2x0 C., particularly from 15 to 50xc2x0 C. and especially between 20 and 30xc2x0 C. The occurrence of vinyl radical polymerization is again inhibited by conducting the reaction under an inert atmosphere, in the dark and in the presence of a radical inhibitor such as 4-methoxyphenol. Typical reaction times are 6-12 hours.
When this step is applied, residual methoxy PEG derivative is completely inactivated and cannot be re-used.
The process of the present invention has the following characteristics:
(a) Free base is the species reacting with the double bond so that the reaction rate will tend to increase as the pH is increased, i.e. as the concentration of free base is increased.
(b) No by-products are normally produced during grafting. The absence of by-product formation means that only unreacted PEG or PEG derivative needs to be separated from the conjugate during the purification stage. Furthermore, it may be possible to re-use the polyethylene glycol compound without further purification. Other PEGylation methods tend to produce toxic by-products which need to be very carefully eliminated.
(c) When the macromolecular material is a compound carrying amino groups, the aminic character of these groups tends to be unaffected by the grafting reaction. This in turn can mean that the electrical nature of the macromolecular material in water does not undergo significant changes as a consequence of grafting. In contrast, many other PEGylation processes cause complete loss of the basic character of amino groups and, therefore, a dramatic change in the electrical nature of the material.
(d) When the macromolecular material is a compound carrying primary amino groups, reaction of these groups with a double bond produces secondary amino groups which may then react with a second double bond to produce a compound comprising two PEG moieties bonded to a single amino nitrogen.
(e) Secondary amine groups tend to react more slowly than primary ones owing to steric hindrance.
The products which are produced using the process of the present invention comprise a coupling moiety or linking group that joins the macromolecular material to the polyethylene glycol compound. When a PEG or PEG derivative activated with an acrylic thioester group is employed, the following coupling moiety is present. 
When a PEG or PEG derivative activated with the acrylic ester group is employed, the following coupling moiety is found. 
Finally, when a PEG or PEG derivative activated with an acrylamido group is employed, the following coupling moiety is found. 
As can be seen from the above formulae, the products contain amido, ester or thioester groups which tend to make the conjugate hydrolysable resulting in the release of the starting PEG or PEG derivative. Such a process of hydrolysis could be advantageous for the biological degradation of the PEG material. It is possible to obtain conjugates with increasing stability towards hydrolysis going from thioester less than ester less than amido. This ability to control the stability of the linkage could be important in some medical applications.
The methoxy PEG (MPEG) derivative whose synthesis is described in Example 1, part (A), has the formula: 
In general, MPEG derivatives having the formula: 
can be prepared by the same procedure as described in Example 1 using, instead of piperazine, another symmetrical bis(primary) or bis(secondary) diamine having the formula: 
wherein
R1 is H or a linear or branched C1-4 alkyl chain; and
R2 is a linear or branched C1-4 alkylene chain.
Alternatively, the group 
may be replaced by the group 
in which R3 and R4 are independently H or a linear or branched C1-3 alkyl chain.
In general, a PEG derivative having the formulae R5"Parenopenst"OCH2CH2"Parenclosest"nY or the formula R5"Parenopenst"OCH2CH2"Parenclosest"nXxe2x80x94Y, where Y is hydroxyl (OH), sulphydryl (SH), primary amino (NH2) or secondary amino (NHR), X is a divalent coupling moiety or linking group whose actual structure depends on the procedure employed for preparing the PEG derivative, n is an integer, e.g. from 10 to 1000, and R5 is alkyl, will be used in the process of the present invention. Preferably n is an integer of from 10 to 100 and R5 is C1-4 alkyl, especially methyl.
The key feature of the PEGylation process of the present invention is the reaction of xe2x80x94NH2, xe2x80x94NHR or xe2x80x94SH groups on the macromolecular material with double bonds on the activated polyethylene glycol compound which are activated by the presence of electron withdrawing groups in the a position.
In principle, the PEGylation process can be applied to molecules containing a polyethylene glycol chain of any molecular weight, but, in practice, molecular weights less than 20 kilodaltons (kD) are preferred, with molecular weights in the range of from 10 to 15 kD being especially preferred.